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Vector-borne pathogens exist in obligate transmission cycles between vector and reservoir host species. Host and vector shifts can lead to geographic expansion of infectious agents and the emergence of new diseases in susceptible individuals. Three bacterial genospecies (Borrelia afzelii, Borrelia bavariensis, and Borrelia garinii) predominantly utilize two distinct tick species as vectors in Asia (Ixodes persulcatus) and Europe (Ixodes ricinus). Through these vectors, the bacteria can infect various vertebrate groups (e.g., rodents, birds) including humans where they cause Lyme borreliosis, the most common vector-borne disease in the Northern hemisphere. Yet, how and in which order the three Borrelia genospecies colonized each continent remains unclear including the evolutionary consequences of this geographic expansion. Here, by reconstructing the evolutionary history of 142 Eurasian isolates, we found evidence that the ancestors of each of the three genospecies probably have an Asian origin. Even so, each genospecies studied displayed a unique substructuring and evolutionary response to the colonization of Europe. The pattern of allele sharing between continents is consistent with the dispersal rate of the respective vertebrate hosts, supporting the concept that adaptation of Borrelia genospecies to the host is important for pathogen dispersal. Our results highlight that Eurasian Lyme borreliosis agents are all capable of geographic expansion with host association influencing their dispersal; further displaying the importance of host and vector association to the geographic expansion of vector-borne pathogens and potentially conditioning their capacity as emergent pathogens., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Spirochetes of the family Borreliaceae are, with one exception, tick-borne pathogens of a variety of vertebrates. The family at present comprises two genera: Borrelia (Swellengrebel), which includes the agents of relapsing fever, avian spirochetosis, and bovine borreliosis, and Borreliella (Gupta et al.), which includes the agents of Lyme disease and was formerly known as 'Borrelia burgdorferi sensulato complex'. The two genera are distinguished not only by their disease associations but also biological features in the tick vector, including tissue location in unfed ticks and transovarial transmission. Borrelia species transmitted by argasid (soft) ticks tend to have more exclusive relationships with their tick vectors than do other Borrelia species and all Borreliella species that have ixodid (hard) ticks as vectors. The division of genera is supported by phylogenomic evidence from whole genomes and by several specific molecular markers. These distinguishing phylogenetic criteria also applied to three new species or isolates of Borrelia that were discovered in ixodid ticks of reptiles, a monotreme, and birds. Although the deep branching of the family from other spirochetes has been a challenge for inferences about evolution of the family, the discovery of related microorganisms in the gut microbiota of other arachnids suggests an ancestral origin for the family as symbionts of ticks and other arachnids., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tick-borne relapsing fever is an infectious disease caused by Borrelia species and are primarily transmitted by Ornithodoros ticks. Prior work indicated that in vitro cultivated spirochetes remain infectious to mice by needle inoculation; however, the impact of laboratory propagation on the pathogens natural life cycle has not been determined. Our current study assessed the effect of serial cultivation on the natural tick-mammalian transmission cycle. First, we evaluated genomic DNA profiles from B. turicatae grown to 30, 60, 120, and 300 generations, and these spirochetes were used to needle inoculate mice. Uninfected nymphal ticks were fed on these mice and acquisition, transstadial maintenance, and subsequent transmission after tick bite was determined. Infection frequencies in mice that were fed upon by ticks colonized with B. turicatae grown to 30, 60, and 120 generations were 100%, 100%, and 30%, respectively. Successful infection of mice by tick feeding was not detected after 120 generations. Quantifying B. turicatae in tick tissues indicated that by 300 generations they no longer colonized the vector. The results indicate that in vitro cultivation significantly affects the establishment of tick colonization and murine infection. This work provides a foundation for the identification of essential genetic elements in the tick-mammalian infectious cycle., Competing Interests: The authors have declared that no competing interests exist.

In 2014, a new tick species, Ixodes inopinatus, was described, which is closely related to Ixodes ricinus. So far, I. inopinatus has been found in Tunisia, Morocco, Spain, Portugal, Romania, Austria, and southern Germany. No data is yet available regarding occurrence of I. inopinatus in northern Germany and the potential role of I. inopinatus as a vector for tick-borne pathogens. Therefore, 3845 DNA samples from Ixodes ticks collected for prevalence studies on Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum during the years 2010-2015 in the northern German cities of Hamburg and Hanover were differentiated into I. ricinus or I. inopinatus by sequencing a part of the 16S rRNA gene. In total, 4% (137/3845) of the sequenced ticks were assigned to the species I. inopinatus and 96% (3708/3845) to I. ricinus. The prevalence of Borrelia spp., Rickettsia spp., and A. phagocytophilum DNA in I. inopinatus was 34% (46/137), 46% (63/137), and 3% (4/137), respectively, whereas the prevalence of these bacteria in I. ricinus was 25% (919/3708), 47% (1729/3708), and 4% (135/3708), respectively. Compared with I. ricinus, significantly more I. inopinatus ticks tested positive for Borrelia. To the best of our knowledge, this is the first report of I. inopinatus in northern Germany. Detection of the DNA of Borrelia spp., Rickettsia spp., and A. phagocytophilum in questing I. inopinatus indicates a potential role of this tick species as a vector of these pathogens, which needs to be confirmed by transmission experiments.

Dermacentor reticulatus is one of the most important European tick species. However, its spatial distribution, seasonality and regional vector role are not well known. This study aimed to gather information about abundance patterns of questing ticks and associated pathogens in unfed female adult D. reticulatus in the Berlin/Brandenburg area. Using the flagging method, questing ticks were collected at four sites in 2010-2012 and 2000 D. reticulatus were analysed regarding infection with Rickettsia, Babesia, Borrelia and Anaplasmataceae by conventional or real-time PCR. Dermacentor reticulatus showed a bimodal activity pattern: highest numbers of adult ticks were recorded between March and end of May (mean 50 ticks/h) and from mid-August until end of November (mean 102 ticks/h). During summer, almost complete inactivity was observed (mean 0.4 ticks/h). Sporadic samplings from December to February revealed tick activity also during winter (mean 47 ticks/h), which was characterised by large fluctuations. Using negative binomial regression analysis, significant influences of the variables sampling site, season and temperature on the abundance of questing D. reticulatus were determined. The parameters relative humidity and year were not of significant importance. PCR analyses showed an average prevalence of 64% for Rickettsia sp. Large differences in pathogen frequencies were observed between sampling sites (31.4-78.3%). Regression analysis demonstrated a significant influence of the sampling site but not of season and year. Examinations regarding other pathogen groups indicated prevalences of 0.25% (Borrelia sp.) and 0.05% (Anaplasmataceae) but absence of Babesia sp. Sequencing of positive samples revealed infections with Rickettsia raoultii, Borrelia miyamotoi, Borrelia afzelii and Anaplasma phagocytophilum. The study shows stable populations of D. reticulatus in Berlin/Brandenburg. People should be aware of ticks throughout the year since Ixodes ricinus is co-endemic and active in spring, summer and autumn while adult D. reticulatus are active throughout the year and even in winter during periods of frost as long as it is warming up during the day. Prevalence of R. raoultii in the present study is among the highest described for D. reticulatus. Borrelia miyamotoi was detected for the first time in D. reticulatus, illustrating the importance of screening studies to evaluate the pathogen structure in D. reticulatus populations., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Vector competence refers to the ability of an arthropod to acquire, maintain, and successfully transmit a microbial pathogen. Tick-borne relapsing fever (TBRF) spirochetes are globally distributed pathogens, and most species are transmitted by argasid ticks of the genus Ornithodoros. A defining characteristic in vector competence is an apparent specificity of a species of TBRF spirochete to a given tick species. In arid regions of the southern United States, Borrelia turicatae is the primary cause of TBRF. Interestingly, there are two populations of the tick vector distributed throughout this region. Ornithodoros turicata is a western population that ranges from California to Texas. There is a gap through Louisiana, Mississippi, and Alabama where the tick has not been identified. An isolated eastern population exists in Florida and was designated a subspecies, O. turicata americanus. A knowledge gap that exists is the poor understanding of vector competence between western and eastern populations of ticks for B. turicatae. In this study, we generated uninfected colonies of O. turicata that originated in Texas and Kansas and of O. turicata americanus. B. turicatae acquisition, maintenance through the molt, and subsequent transmission were evaluated. Our findings revealed significant differences in murine infection after feeding infected O. turicata and O. turicata americanus ticks on the animals. Interestingly, the salivary glands of both tick populations were colonized with B. turicatae to similar densities. Our results suggest that the salivary glands of the tick colonies assessed in this study impact vector competence of the evaluated B. turicatae isolates. IMPORTANCE Several knowledge gaps exist in the vector competence of various geographical populations of O. turicata that transmit B. turicatae A western population of this tick is distributed from California to Texas, and an eastern population exists in Florida. Utilizing western and eastern populations of the vector, we studied acquisition and transmission of two B. turicatae isolates. Regardless of the isolate used, infection frequencies were poor in mice after the eastern population feeding on them. Since salivary gland colonization is essential for B. turicatae transmission, these tissues were further evaluated. Interestingly, the salivary glands from the two populations were similarly colonized with B. turicatae. These findings suggest the role of tick saliva in the establishment of infection and that the salivary glands may be a bottleneck for successful transmission., (Copyright © 2018 American Society for Microbiology.)

Endemic tick-borne relapsing fever (TBRF) has not been documented in Nigeria, yet clinically compatible cases have been described, and soft tick species are endemic in surrounding countries. Consequently, our aim was to investigate if TBRF-associated Borrelia is present in Nigeria. To address this, we examined 49 soft tick pools to identify the tick species and to screen for Borrelia The tick species was revealed by 16S rRNA gene amplification and Sanger sequencing to be Ornithodoros savignyi , an aggressive, multihost, rapidly feeding species with significant veterinary impact. We detected a Borrelia organism in 3 of 49 pooled samples (6%). Molecular analysis of amplified 16S rRNA and flagellin genes and intragenic spacer fragments disclosed that this Borrelia organism was synonymous with the recently described organism " Candidatus Borrelia kalaharica," identified in a tourist returning to Germany from South Africa. Given the widespread endemic range of this tick vector, TBRF should be considered part of the differential diagnosis for patients with fever returning from arid areas of Africa and further afield., (Copyright © 2018 American Society for Microbiology.)

Lemurs are primate species that are endemic to Madagascar. At present, about 90% of lemur species are endangered, and 5 species are among the 25 most endangered primates worldwide. Health status is a major factor impacting the viability of wild populations of many endangered species including lemurs. Given this context, we analyzed the microbiome of 24 specimens of Haemaphysalis lemuris, the most common tick parasitizing lemurs in their native habitats. Ticks were collected from 6 lemur species and microbiomes analyzed using next-generation sequencing. Our results show that the H. lemuris microbiome is highly diverse, including over 500 taxa, 267 of which were identified to genus level. Analysis of the microbiome also shows that there is a distinct "host" (lemur species) component when explaining the differences among and between microbial communities of H. lemuris. This "host" component seems to overwhelm any "locality" (geographic origin of the sample) component. In addition to the microbiome data, targeted PCR was used to test for the presence of three pathogens recently detected in the blood of wild lemurs: Borrelia sp., Candidatus Neoehrlichia sp., and Babesia sp. Overall, the presence of DNA of Rickettsia spp., Bartonella spp., Francisella spp., and a Babesia sp., in H. lemuris, is consistent with the hypothesis that these ectoparasites may act as vector for these pathogens. Further studies assessing vector competence are needed to confirm this hypothesis., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flaB sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tick-borne relapsing fever in western North America is a zoonosis caused by the spirochete bacterium, Borrelia hermsii, which is transmitted by the bite of infected Ornithodoros hermsi ticks. The pathogen is maintained in natural cycles involving small rodent hosts such as chipmunks and tree squirrels, as well as the tick vector. In order for these ticks to establish sustained and viable populations, a narrow set of environmental parameters must exist, primarily moderate temperatures and moderate to high amounts of precipitation. Maximum Entropy Species Distribution Modeling (Maxent) was used to predict the species distribution of O. hermsi and B. hermsii through time and space based on current climatic trends and future projected climate changes. From this modeling process, we found that the projected current distributions of both the tick and spirochete align with known endemic foci for the disease. Further, global climate models predict a shift in the distribution of suitable habitat for the tick vector to higher elevations. Our predictions are useful for targeting surveillance efforts in areas of high risk in western North America, increasing the efficiency and accuracy of public health investigations and vector control efforts.

Ixodes ticks transmit Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis (LB). These tick species also transmit Borrelia miyamotoi, which was recently found to cause infections in humans. We were interested in the prevalence of B. miyamotoi infection in ticks and natural hosts in The Netherlands, and to what extent ticks are co-infected with B. burgdorferi. In addition, erythema migrans has been sporadically described in B. miyamotoi-infected patients, but these skin lesions might as well represent co-infections with B. burgdorferi s.l. We therefore investigated whether B. miyamotoi was present in LB-suspected skin lesions of patients referred to our tertiary Lyme disease clinic. 3360 questing Ixodes ricinus nymphs as well as spleen tissue of 74 rodents, 26 birds and 10 deer were tested by PCR for the presence of B. miyamotoi. Tick lysates were also tested for the presence of B. burgdorferi s.l. Next, we performed a PCR for B. miyamotoi in 31 biopsies from LB-suspected skin lesions in patients visiting our tertiary Lyme center. These biopsies had been initially tested for B. burgdorferi s.l. by PCR, and the skin lesions had been investigated by specialized dermatologists. Out of 3360 unfed (or questing) nymphs, 313 (9.3%) were infected with B. burgdorferi s.l., 70 (2.1%) were infected with B. miyamotoi, and 14 (0.4%) were co-infected with B. burgdorferi s.l. and B. miyamotoi. Co-infection of B. burgdorferi s.l. with B. miyamotoi occurred more often than expected from single infection prevalences (p=0.03). Both rodents (9%) and birds (8%) were found positive for B. miyamotoi by PCR, whereas the roe deer samples were negative. Out of 31 LB-suspected skin biopsies, 10 (32%) were positive for B. burgdorferi s.l. while none were positive for B. miyamotoi. The significant association of B. burgdorferi s.l. with B. miyamotoi in nymphs implies the existence of mutual reservoir hosts. Indeed, the presence of B. miyamotoi DNA indicates systemic infections in birds as well as rodents. However, their relative contributions to the enzootic cycle of B. miyamotoi requires further investigation. We could not retrospectively diagnose B. miyamotoi infection using biopsies of LB-suspected skin lesions, supporting the hypothesis that B. miyamotoi is not associated with LB-associated skin manifestations. However, this warrants further studies in larger sets of skin biopsies. A prospective study focused on acute febrile illness after a tick bite could provide insight into the incidence and clinical manifestations of B. miyamotoi infection in The Netherlands., (Copyright © 2016 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

We evaluated Amblyomma americanum (lone star tick) and Dermacentor variabilis (American dog tick) in northeast Missouri for the presence of Borrelia, Ehrlichia, and Rickettsia bacteria. We collected actively questing ticks from four sites within Adair County, Missouri. A total of 15,162 ticks were collected, of which 13,980 were grouped in 308 pools (lone star ticks, 288 pools; American dog ticks, 20 pools) and tested for presence/absence of bacteria using polymerase chain reaction. Infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of the 308 pools tested, 229 (74.4%) were infected with bacteria and the overall MLE of the infection rate per 100 ticks was calculated as 2.9% (CI 2.61-3.21). Infection rates varied among life stages, 28.6% (CI 23.89-33.97) in adults, 7.0% (CI 5.10-9.86) in nymphs, and 1.0% (CI 0.75-1.20) in larvae. In the 116 adult lone star pools, infection rates were calculated for Borrelia lonestari (1.4%), Borrelia spp. (2.7%), Ehrlichia chaffeensis (6.1%), Ehrlichia ewingii (3.3%), Rickettsia amblyommii (18.3%), and Rickettsia montanensis (0.4%). Infection rates for the 52 nymphal lone star pools were calculated as B. lonestari (1.03%), Borrelia spp. (0.40%), E. chaffeensis (2.02%), E. ewingii (0.24%), and R. amblyommii (2.70%). In the 20 adult American dog tick pools, infection rates were determined as E. chaffeensis (9.47%), E. ewingii (5.47%), and R. montanensis (8.06%). Eight Borrelia samples were sequenced with five 99-100% identical to B. burgdorferi (s.l.) and three 99% identical to B. lonestari. Eight samples were sequenced for E. chaffeensis (all 99-100% identical) and one sample was sequenced for E. ewingii (99% identical). Seven samples were sequenced for Rickettsia and three were 99% identical to R. montanensis and four were 100% identical to R. amblyommii. This study demonstrates B. lonestari, E. chaffeensis, E. ewingii, R. amblyommii, and R. montanensis in northeast Missouri ticks for the first time. Understanding the presence and epidemiology of these causative (E. chaffeensis and E. ewingii) and suspected (B. lonestari and R. amblyommii) agents in Missouri should increase awareness of potential tick-borne disease in the medical community., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Due to an increasing diversity of bacterial pathogens known to be transmitted by hard ticks (Acari: Ixodidae) in North America, a comprehensive assay is needed to detect and differentiate among these numerous tick-borne pathogens. We describe an expanded protocol using a combination of multiplex polymerase chain reaction and reverse line blot hybridization to detect a greater diversity of infectious agents than were previously detectable. Ten novel oligonucleotide probes, either individually or in concert, enabled or enhanced identification of six Borrelia species, three Rickettsia species, and one Ehrlichia species. Simultaneous detection of these numerous tick-borne pathogens can advance surveillance efforts and improve accuracy of detection and, thus, reporting., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Lyme borreliosis is the most prevalent tick-borne disease in Europe. It is caused by spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex and transmitted to humans by ticks of the genus Ixodes. Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana are the most common genospecies in Central Europe. In contrast, Borrelia lusitaniae predominates in Mediterranean countries such as Portugal, Morocco, and Tunisia. In Slovakia, its prevalence is low and restricted to only a few sites. The aim of our research was to study the expansion of ticks into higher altitudes in the ecosystem of the Malá Fatra mountains (north Slovakia) and their infection with B. burgdorferi s.l. pathogens. Questing ticks were collected by flagging in seven years (2004, 2006-2011) at three different altitudes: low (630-660 m above sea level (ASL)), intermediate (720-750 m ASL), and high (1040-1070 m ASL). Tick abundance was highest at the lowest altitude and lowest at the highest altitude. The average infection prevalence of B. burgdorferi s.l. in nymphs and adults was 16.8% and 36.2%, respectively. The number of infected ticks decreased from 38.5% at the lowest altitude to 4.4% at the highest altitude. B. lusitaniae was the most frequently found genospecies (>60% of the ticks found positive for B. burgdorferi s.l.) in all sites in all the studied years with the exception of 2008 when B. afzelii predominated (62%). Our study confirms the spread of Ixodes ricinus ticks to higher altitudes in Slovakia. The discovery that our mountain study sites were a natural foci of B. lusitaniae was unexpected because this genospecies is usually associated with lizards and xerothermic habitats., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Little is known about the role of endemic ticks as vectors for bacterial and protozoan pathogens for animals and humans in Madagascar and their interaction in anthropogenic habitats where humans, their livestock and native Malagasy species (vectors and hosts) come into more frequent contact than in natural forest ecosystems. The aims of the study were (1) to test whether habitat degradation is associated with increased infestation of tortoises by ticks and (2) to investigate whether ticks carried Babesia, Borrelia or Rickettsia species that might be pathogenic for humans and livestock. We studied hard ticks of two endemic Malagasy tortoises, Astrochelys radiata and Pyxis arachnoides in March and April 2013 in southwest Madagascar. Two tortoise habitats were compared, the National Park of Tsimanampetsotsa and the adjacent degraded pasture and agricultural land at the end of the wet season. Ticks were screened for protozoan and bacterial pathogens via PCR on DNA isolated from ticks using genus-specific primers. Only one out of 42 A. radiata collected from both habitats had ticks. The low prevalence did not allow further analyses of the effect of habitat degradation. Forty-two P. arachnoides were found in the anthropogenic habitat and 36 individuals in the national park. Tick infestation rates of P. arachnoides differed significantly between the two study sites. Tortoises inside the park had lower tick prevalence than outside (8 of 36 (22%) versus 32 of 42 individuals (76%)) and infected animals tended to have fewer ticks inside than outside the park. All ticks collected in both habitats were adults of the ixodid tick Amblyomma chabaudi, which is supposed to be a host-specific tick of P. arachnoides. Screening for Borrelia sp. and Babesia sp. was negative in all ticks. But all A. chabaudi ticks were infected with Rickettsia africae, known to cause spotted fever in humans. Thus, habitat degradation seems to be linked to higher infestation of tortoises with ticks with possible consequences for humans and their livestock., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Adult Ixodes persulcatus were collected in highly populated districts in Irkutsk city, Russia, and in popular recreational and professional areas in its neighboring territories. Borrelia miyamotoi infection in I. persulcatus was examined using multiplex Taqman-PCR targeting 16S rDNA, and nested PCR and sequencing analyses targeting flaB and 16S rDNA. B. miyamotoi and Lyme disease Borrelia species were detected in 13 (infection rate, 2.9%) and 77 (17.3%) out of 445 I. persulcatus ticks, respectively, collected from 4 sites around the Baikal Lake. The 16S rDNA and flaB sequences of these amplicons were closely related to those of B. miyamotoi detected and/or isolated from I. persulcatus in Japan and Far Eastern Russia, and clustered separately from those of Europe and North America. These results indicate that additional surveillance for B. miyamotoi infection is needed in order to determine how it affects human health in Irkutsk City and its neighboring territories., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

To describe the presence and distribution of tickborne bacteria and their vectors in Texas, USA, we screened ticks collected from humans during 2008-2014 for Rickettsia, Borrelia, and Ehrlichia spp. Thirteen tick species were identified, and 23% of ticks carried bacterial DNA from at least 1 of the 3 genera tested.

Background: Our study aimed to assess the presence of different pathogens in ticks collected in two regions in Côte d'Ivoire., Methodology/principal Findings: Real-time PCR and standard PCR assays coupled to sequencing were used. Three hundred and seventy eight (378) ticks (170 Amblyomma variegatum, 161 Rhipicepalus microplus, 3 Rhipicephalus senegalensis, 27 Hyalomma truncatum, 16 Hyalomma marginatum rufipes, and 1 Hyalomma impressum) were identified and analyzed. We identified as pathogenic bacteria, Rickettsia africae in Am. variegatum (90%), Rh. microplus (10%) and Hyalomma spp. (9%), Rickettsia aeschlimannii in Hyalomma spp. (23%), Rickettsia massiliae in Rh. senegalensis (33%) as well as Coxiella burnetii in 0.2%, Borrelia sp. in 0.2%, Anaplasma centrale in 0.2%, Anaplasma marginale in 0.5%, and Ehrlichia ruminantium in 0.5% of all ticks. Potential new species of Borrelia, Anaplasma, and Wolbachia were detected. Candidatus Borrelia africana and Candidatus Borrelia ivorensis (detected in three ticks) are phylogenetically distant from both the relapsing fever group and Lyme disease group borreliae; both were detected in Am. variegatum. Four new genotypes of bacteria from the Anaplasmataceae family were identified, namely Candidatus Anaplasma ivorensis (detected in three ticks), Candidatus Ehrlichia urmitei (in nine ticks), Candidatus Ehrlichia rustica (in four ticks), and Candidatus Wolbachia ivorensis (in one tick)., Conclusions/significance: For the first time, we demonstrate the presence of different pathogens such as R. aeschlimannii, C. burnetii, Borrelia sp., A. centrale, A. marginale, and E. ruminantium in ticks in Côte d'Ivoire as well as potential new species of unknown pathogenicity.

Borrelia miyamotoi, a relapsing fever spirochete, has been found recently in Ixodes ricinus ticks; however, little is known about its spatial distribution and potential local impact on human health. A total of 640 ticks (447 nymphs and 193 adults) collected throughout Portugal were analyzed using two nested PCR protocols, one targeting the flagellin gene and the other the internal transcribed space region between the 5S and the 23S rRNA. As a result, B. miyamotoi was detected, for the first time, in one guesting I. ricinus nymph collected in the Lisboa district. In addition, a prevalence of 11% (71/640) for B. burgdorferi sensu lato was obtained. Even though no human relapsing fever cases due to infection by B. miyamotoi have been reported yet in Portugal, surveillance must be improved to provide better insight into the prevalence and distribution of this spirochete in ticks.

Background: The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks., Methods: Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont "Candidatus Midichloria mitochondrii" (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM., Results: Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel "Candidatus Neoehrlichia" spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai., Conclusions: Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel "Candidatus Neoehrlichia" spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

Borrelia bavariensis is a recently described agent of Lyme disease within the B. burgdorferi sensu lato species complex and exhibits a strong capacity for human pathogenicity. B. bavariensis strains are widely distributed in Eurasia spanning the distribution range of the tick vectors Ixodes persulcatus and I. ricinus. It has been suggested that B. bavariensis forms two populations, one of which arose through vector adaptation and geographic expansion. We have performed phylogenetic and population genetic analyses with next-generation sequencing data of 26 strains of B. bavariensis targeting the main linear chromosome and two plasmids (lp54, cp26). A very low number of single nucleotide polymorphisms (SNPs) was found in the European population and a deep branching pattern between European and Asian B. bavariensis was observed in all phylogenies. The results confirm the population structure of B. bavariensis and strongly support the hypothesis of clonal expansion of the European population of B. bavariensis. In addition, signals of positive selection identified in the populations further support the hypothesis that the European population of B. bavariensis likely underwent vector adaptation in its recent evolutionary history. Identified genes represent promising candidates for experimental vector adaptation studies. Thus, this species forms a very good model to study vector adaptation, which is known to play an important role in the geographic distribution of B. burgdorferi. Analysis of well known virulence determinants that are attributed to severity of clinical manifestation in B. burgdorferi s.s. revealed no variation within the European population of B. bavariensis, underlining the importance of including various Borrelia species into investigations that aim to understand the pathogenesis of Lyme disease agents., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Long-term studies on natural foci of ixodid tick-borne borrelioses (ITBB) have been performed in Chusovskoi district of Perm region, the Middle Urals, where the vectors of these infections are represented by two ixodid tick species: the taiga tick Ixodes persulcatus and many times less abundant vole tick I. trianguliceps. Over 10 years, more than 6000 half-engorged ticks were collected from small forest mammals using the standard procedure, and 1027 I. persulcatus and 1142 I. trianguliceps ticks, individually or in pools, were used to inoculate BSK-2 medium. As a result, 199 Borrelia isolates were obtained. Among them, 177 isolates were identified, and the rrf(5S)-rrl(23S) intergenic spacer sequence was determined in 57 isolates. The prevalence of Borrelia infection in I. persulcatus larvae and nymphs averaged 31.0 and 53.3%, while that in I. trianguliceps larvae, nymphs, and adult ticks was five to ten times lower: 2.6, 10.2, and 8.1%, respectively. Each of the two tick species was found to carry both ITBB agents circulating in the Middle Ural foci (Borrelia garinii and B. afzelii), but the set of genogroups and genovariants of these spirochetes in I. trianguliceps proved to be far less diverse. According to the available data, this tick, compared to I. persulcatus, is generally less susceptible to Borrelia infection (especially by B. afzelii). Taking into account of its relatively low abundance, it appears that I. trianguliceps cannot seriously influence the course of epizootic process in ITBB foci of the study region, whereas highly abundant I. persulcatus with the high level of Borrelia infection is obviously a key component of these parasitic systems. A similar situation may well be typical for the entire geographic range shared by the two tick species., (Copyright © 2015. Published by Elsevier GmbH.)

Little is known about Borrelia species transmitted by hard ticks in Ethiopia. The present study was conducted from November 2011 through March 2014 to address the occurrence and molecular identity of these bacteria in ixodid ticks infesting domestic animals in Oromia, Ethiopia. A total of 767 ixodid ticks collected from domestic animals were screened for Borrelia DNA by quantitative (q) real-time PCR followed by standard PCR and sequencing to identify the species. Overall, 3.8% (29/767) of the tested ticks were positive for Borrelia DNA, including 8/119 (6.7%) Amblyomma cohaerens, 1/42 (2.4%) Am. gemma, 3/53 (5.7%) Am. variegatum, 5/22 (22.7%) Amblyomma larvae, 3/60 (5%) Amblyomma nymphs, 2/139 (1.4%) Rhipicephalus (Boophilus) decoloratus, 2/31 (6.4%) Rh. decoloratus nymphs, and 5/118 (4.2%) Rh. pulchellus using 16S genus-specific qPCR. The prevalence of Borrelia DNA was significantly higher in genus Amblyomma (20/298, 6.7%) than in the genus Rhipicephalus (9/417, 2.1%) ticks (P=0.001). Sequencing of PCR products from the flaB and 16S rRNA genes of Borrelia spp. from Amblyomma ticks showed the presence of a new species between the relapsing fever and Lyme disease groups. However, Borrelia sp. detected in Rhipicephalus ticks clustered with B. theileri/B. lonestari. The human pathogenicity of the Borrelia sp. detected in Amblyomma ticks from Ethiopia has not yet been investigated, whereas the Borrelia sp. detected in Rhipicephalus ticks in our study is the causative agent of bovine borreliosis in cattle and may have veterinary importance in different parts of Ethiopia. Furthermore, the detection of previously unrecognized Borrelia species in Amblyomma and Rhipicephalus ticks in Ethiopia generates additional questions concerning the bacterial fauna in hard ticks and will prompt researchers to perform detailed studies for better understanding of ixodid ticks associated bacteria., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Ixodid ticks play an important role in the transmission and ecology of infectious diseases. Information about the circulation of tick-borne bacteria in ticks is lacking in Ecuador. Our aims were to investigate the tick species that parasitize Andean tapirs and cattle, and those present in the vegetation from the buffer zone of the Antisana Ecological Reserve and Cayambe-Coca National Park (Ecuador), and to investigate the presence of tick-borne bacteria., Methods: Tick species were identified based on morphologic and genetic criteria. Detection of tick-borne bacteria belonging to Rickettsia, Anaplasma, Ehrlichia and Borrelia genera was performed by PCRs., Results: Our ticks included 91 Amblyomma multipunctum, 4 Amblyomma spp., 60 Rhipicephalus microplus, 5 Ixodes spp. and 1 Ixodes boliviensis. A potential Candidatus Rickettsia species closest to Rickettsia monacensis and Rickettsia tamurae (designated Rickettsia sp. 12G1) was detected in 3 R. microplus (3/57, 5.3%). In addition, Anaplasma spp., assigned at least to Anaplasma phagocytophilum (or closely related genotypes) and Anaplasma marginale, were found in 2 A. multipunctum (2/87, 2.3%) and 13 R. microplus (13/57, 22.8%)., Conclusions: This is the first description of Rickettsia sp. in ticks from Ecuador, and the analyses of sequences suggest the presence of a potential novel Rickettsia species. Ecuadorian ticks from Andear tapirs, cattle and vegetation belonging to Amblyomma and Rhipicephalus genera were infected with Anaplasmataceae. Ehrlichia spp. and Borrelia burgdorferi sensu lato were not found in any ticks.

Borrelia bavariensis was recently described as a distinct genospecies among the B. burgdorferi sensu lato complex. The prevalence of B. bavariensis in Austria, a highly endemic area for tick-transmitted pathogens, is scarcely characterized. To investigate the prevalence of B. bavariensis in Ixodes ricinus ticks we reevaluated the results of a study conducted in 518 ticks from southeastern Austria collected in 2002 and 2003. The presence of B. burgdorferi s.l.-specific DNA in ticks was analyzed by a PCR for the outer surface protein A (ospA) gene. Borrelia species were differentiated by restriction fragment length polymorphism (RFLP) analysis, and samples positive for B. bavariensis were further analyzed by multilocus sequence analysis. Two of 133 (1.5%) B. burgdorferi s.l.-positive I. ricinus ticks were infected with B. bavariensis. Both specimens were coinfected with the OspA serotype 5 of B. garinii. Borrelia bavariensis is present; however, seem to be rare in I. ricinus ticks in southeastern Austria., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

A relapsing fever Borrelia sp. similar to Borrelia lonestari (herein referred to as B. lonestari-like) was detected from wild sika deer (Cervus nippon yesoensis) and Haemaphysalis ticks in the eastern part of Hokkaido, Japan. The total prevalence of this Borrelia sp. in tested deer blood samples was 10.6% using conventional PCR and real-time PCR. The prevalence was significantly higher in deer fawns compared to adults (21.9% and 9.4%, respectively). Additionally, there was significant regional difference between our two sampling areas, Shiretoko and Shibetsu with 17% and 2.8% prevalence, respectively. Regional differences were also found in tick species collected from field and on deer. In the Shiretoko region, Haemaphysalis spp. were more abundant than Ixodes spp., while in Shibetsu, Ixodes spp. were more abundant. Using real-time PCR analysis, B. lonestari-like was detected from 2 out of 290 adult Haemaphysalis spp. ticks and 4 out of 76 pools of nymphs. This is the first report of a B. lonestari-like organism in Haemaphysalis spp. ticks, and the first phylogenetic analysis of this B. lonestari-like organism in Asia. Based on our results, Haemaphysalis spp. are the most likely candidates to act as a vector for B. lonestari-like; furthermore, regional variation of B. lonestari-like prevalence in sika deer may be dependent on the population distribution of these ticks., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Ixodes ricinus is the primary vector of Borrelia spirochetes in Europe, including both the Lyme borreliosis (LB) group and the relapsing fever (RF) group. The aim of the study was to estimate the prevalence of different genospecies from the B. burgdorferi s.l. complex and B. miyamotoi in questing I. ricinus collected in chosen areas in Lower Silesia, SW Poland. A total of 599 I. ricinus ticks were investigated using the PCR-RFLP method. The calculated overall minimum infection rate of ticks with Borrelia spirochetes in Lower Silesia was 15.5%. Five different restriction patterns, characteristic of B. afzelii, B. garinii, B. burgdoreri s.s., B. valaisiana, and B. miyamotoi, were obtained and confirmed by DNA sequencing. At least 14% of ticks were infected with B. burgdorferi s.l. while B.afzelii was the dominant genospecies (68.5%). The MIR for B. miyamotoi was calculated at 2%. Four co-infections in single adult ticks were found: B. miyamotoi/B. afzelii, B. miyamotoi/B. burdorferi s.s., B. miyamotoi/B. garinii, and B. afzelii/B. burgdorferi s.s. The results of this study confirm the risk of LB and RF occuring in both urban and protected areas., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The diversity of Lyme disease (LD) and relapsing fever (RF)-group spirochetes in the metropolitan San Francisco Bay area in northern California is poorly understood. We tested Ixodes pacificus, I. spinipalpis, and small mammals for presence of borreliae in Alameda County in the eastern portion of San Francisco Bay between 2009 and 2012. Analyses of 218 Borrelia burgdorferi sensu lato (Bb sl) culture or DNA isolates recovered from host-seeking I. pacificus ticks revealed that the human pathogen Bb sensu stricto (hereinafter, B. burgdorferi) had the broadest habitat distribution followed by B. bissettii. Three other North American Bb sl spirochetes, B. americana, B. californiensis and B. genomospecies 2, also were detected at lower prevalence. OspC genotyping of the resultant 167 B. burgdorferi isolates revealed six ospC alleles (A, D, E3, F, H and K) in I. pacificus. A novel spirochete belonging to the Eurasian Bb sl complex, designated CA690, was found in a questing I. spinipalpis nymph. Borrelia miyamotoi, a relapsing-fever (RF) group spirochete recently implicated as a human pathogen, was detected in 24 I. pacificus. Three rodent species were infected with Bb sl: the fox squirrel (Sciurus niger) with B. burgdorferi, and the dusky-footed wood rat (Neotoma fuscipes) and roof rat (Rattus rattus) with B. bissettii. Another spirochete that clustered phylogenetically with the Spanish R57 Borrelia sp. in a clade distinct from both the LD and RF groups infected some of the roof rats. Together, eight borrelial genospecies were detected in ticks or small mammals from a single Californian county, two of which were related phylogenetically to European spirochetes., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Recent reports suggest a potential for transmission of a newly discovered rickettsial endosymbiont, Midichloria mitochondrii, to animals and humans from feeding ticks (Ixodes ricinus). Using molecular methods; I. ricinus, sheep and red deer in Anaplasma phagocytophilum-endemic areas of Norway, were examined to see if they were infected by M. mitochondrii or related organisms like Wolbachia pipientis and Rickettsia spp. A total of 532 ticks collected from pastures, 76 blood samples from grazing lambs and 12 organ samples from hunted deer, were analyzed during the study. All larval pools, 60.4% pooled nymphs and 35.1% of adult ticks were positive for M. mitochondrii. There was a significant difference between geographical areas in the prevalence of M. mitochondrii infection among nymphs. A total of 2.2% pooled nymphs and 5.3% adult ticks were positive for A. phagocytophilum. Eleven percent of pooled nymphs were positive for Borrelia spp, 2.2% of pooled nymphs and 3.5% of adult ticks were positive for Rickettsia spp. and none of the ticks were positive for W. pipientis. The prevalence of A. phagocytophilum infection was 54% and 75% in grazing lambs and deer, respectively. No animals were positive for Borrelia spp., M. mitochondrii, Rickettsia spp. or W. pipientis. The reported findings suggest that M. mitochondrii is widespread in tick populations at different geographical sites, and may appear in co-infection with A. phagocytophilum, Borrelia spp. and Rickettsia spp. in ticks., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Ticks are important vector arthropods of human and animal pathogens. As information about agents of disease circulating in vectors in Portugal is limited, the aim of the present study was to detect bacteria and parasites with veterinary and zoonotic importance in ticks collected from dogs, cats, and field vegetation. A total of 925 ticks, comprising 888 (96.0%) adults, 8 (0.9%) nymphs, and 29 (3.1%) larvae, were collected in 4 geographic areas (districts) of Portugal. Among those, 620 (67.0%) were removed from naturally infested dogs, 42 (4.5%) from cats, and 263 (28.4%) were questing ticks obtained from field vegetation. Rhipicephalus sanguineus was the predominant tick species, and the only one collected from dogs and vegetation, while all Ixodes ricinus specimens (n=6) were recovered from cats. Rickettsia massiliae and Rickettsia conorii were identified in 35 ticks collected from cats and dogs and in 3 ticks collected from dogs. Among ticks collected from cats or dogs, 4 Rh. sanguineus specimens were detected with Hepatozoon felis, 3 with Anaplasma platys, 2 with Hepatozoon canis, one with Anaplasma phagocytophilum, one with Babesia vogeli, one with Borrelia burgdorferi sensu lato and one with Cercopithifilaria spp. Rickettsia helvetica was detected in one I. ricinus tick collected from a cat. To the best of our knowledge, this was the first time that Cercopithifilaria spp., Ba. vogeli, H. canis, and H. felis have been detected in ticks from Portugal. The wide range of tick-borne pathogens identified, some of zoonotic concern, suggests a risk for the emergence of tick-borne diseases in domestic animals and humans in Portugal. Further studies on these and other tick-borne agents should be performed to better understand their epidemiological and clinical importance, and to support the implementation of effective control measures., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: In France as elsewhere in Europe the most prevalent TBD in humans is Lyme borreliosis, caused by different bacterial species belonging to Borrelia burgdorferi sensu lato complex and transmitted by the most important tick species in France, Ixodes ricinus. However, the diagnosis of Lyme disease is not always confirmed and unexplained syndromes occurring after tick bites have become an important issue. Recently, B. miyamotoi belonging to the relapsing fever group and transmitted by the same Ixodes species has been involved in human disease in Russia, the USA and the Netherlands. In the present study, we investigate the presence of B. miyamotoi along with other Lyme Borreliosis spirochetes, in ticks and possible animal reservoirs collected in France., Methods: We analyzed 268 ticks (Ixodes ricinus) and 72 bank voles (Myodes glareolus) collected and trapped in France for the presence of DNA from B. miyamotoi as well as from Lyme spirochetes using q-PCR and specific primers and probes. We then compared the French genotypes with those found in other European countries., Results: We found that 3% of ticks and 5.55% of bank voles were found infected by the same B. miyamotoi genotype, while co-infection with other Lyme spirochetes (B. garinii) was identified in 12% of B. miyamotoi infected ticks. Sequencing showed that ticks and rodents carried the same genotype as those recently characterized in a sick person in the Netherlands., Conclusions: The genotype of B. miyamotoi circulating in ticks and bank voles in France is identical to those already described in ticks from Western Europe and to the genotype isolated from a sick person in The Netherlands. This results suggests that even though no human cases have been reported in France, surveillance has to be improved. Moreover, we showed that ticks could simultaneously carry B. miyamotoi and Lyme disease spirochetes, increasing the problem of co-infection in humans.

In Europe, in addition to Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus, other zoonotic pathogens, like B. miyamotoi, a species related to the relapsing fever spirochaetes, Candidatus Neoehrlichia mikurensis (N. mikurensis), Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum have been reported in the ixodid tick Ixodes ricinus. No study was conducted to identify reservoir hosts for these pathogens. Here, we investigated the role played by wild rodents in the natural transmission cycle of B. miyamotoi, N. mikurensis, R. helvetica, R. monacensis, and A. phagocytophilum in Switzerland. In 2011 and 2012, small mammals were captured in an area where these pathogens occur in questing ticks. Ixodes ricinus ticks infesting captured small mammals were analysed after their moult by PCR followed by reverse line blot to detect the different pathogens. Xenodiagnostic larvae were used to evaluate the role of rodents as reservoirs and analysed after their moult. Most of the 108 captured rodents (95.4%) were infested by I. ricinus ticks; 4.9%, 3.9%, 24.0%, and 0% of the rodents were infested by Borrelia, N. mikurensis, Rickettsia spp., and A. phagocytophilum-infected larvae, respectively. Borrelia afzelii, B. miyamotoi, N. mikurensis, Rickettsia spp., and A. phagocytophilum were detected in 2.8%, 0.17%, 2.6%, 6.8%, and 0% of the ticks attached to rodents, respectively. Borrelia afzelii was transmitted by 4 rodents to 41.2% of the xenodiagnostic ticks, B. miyamotoi by 3 rodents to 23.8%, and N. mikurensis was transmitted by 6 rodents to 41.0% of the xenodiagnostic ticks. None of the tested rodent transmitted Rickettsia spp. or A. phagocytophilum to I. ricinus xenodiagnostic larvae. This study showed that rodents are reservoir hosts for B. miyamotoi and N. mikurensis in Europe., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Migratory birds act as hosts and long-distance vectors for several tick-borne infectious agents. Here, feeding Ixodes ticks were collected from migratory birds during the autumn migration period in Latvia and screened for the presence of epidemiologically important non-viral pathogens. A total of 93 DNA samples of ticks (37 larvae and 56 nymphs) removed from 41 birds (order Passeriformes, 9 species) was tested for Lyme borreliosis spirochaetes, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp. Borrelia burgdorferi DNA was detected in 18% of the tick samples, and a majority of infected ticks were from thrush (Turdus spp.) birds. Among the infected ticks, Borrelia valaisiana was detected in 41% of cases, Borrelia garinii in 35%, and mixed Bo. valaisiana and Bo. garinii infection in 24%. Anaplasma phagocytophilum DNA was detected in 2% of ticks, R. helvetica in 12%, and Babesia spp. pathogens in 4% of ticks. Among these samples, 3 Babesia species were identified: Ba. divergens, Ba. microti, and Ba. venatorum. Coinfection with different pathogens that included mixed infections with different Borrelia genospecies was found in 20% of nymphal and 3% of larval Ixodes ticks. These results suggest that migratory birds may support the circulation and spread of medically significant zoonoses in Europe., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Objectives: Over a 3-y period, Ixodes ricinus ticks were randomly collected to study the prevalence of 4 Borrelia species: B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. valaisiana. While B. burgdorferi s. s., B. afzelii, and B. garinii have been associated with human borreliosis in Norway for several years, B. valaisiana was reported in a Norwegian tick for the first time in 2010., Methods: A real-time polymerase chain reaction (qPCR) was developed as an easy-to-use method, with high sensitivity and specificity, to detect and genospecies-type B. burgdorferi s. s., B. afzelii, B. garinii, and B. valaisiana in I. ricinus ticks. A combination of species-specific primers and TaqMan MGB probes labelled with fluorescents with different emission spectra, ensured a highly specific method with the potential to detect more than 1 genospecies in 1 run. Sequencing of the housekeeping gene recG from 48 Borrelia-positive samples was used to confirm specificity. Denaturing gradient gel electrophoresis profiling of tick-borne bacteria was used to help optimize the assay sensitivity., Results: The qPCR assay was applied to analyze 1808 I. ricinus ticks collected in the field, which resulted in an overall infection rate of 14.8%, 18.7%, and 14.3% in 2010, 2011, and 2012, respectively. The majority of the Borrelia-infected ticks were infected with B. afzelii., Conclusions: The overall infection rate is somewhat lower than that reported in the most recent study of the infection rate in southern Norway in 2010, and this study indicates that the infection rate varies from one year to another.

Little is known about the eco-epidemiology of Lyme disease in southern California, a region where the incidence is much lower than it is in northern California. Here, we sought to discover the previously unknown microhabitats of nymphs of the primary vector, the western black-legged tick (Ixodes pacificus), in 3 moderately to heavily-utilized state parks in the Santa Monica Mountains in Los Angeles County; to elucidate the seasonal distribution and abundance of adults of I. pacificus and another human-biter, the Pacific Coast tick (Dermacentor occidentalis); and to determine what Lyme-disease or relapsing-fever group borreliae are present in questing nymphs or adult ticks. I. pacificus nymphs were collected infrequently at various times of day in 2 chaparral or 7 woodland litter areas by dragging (combined mean=0.4 nymphs per hour). The western fence lizard (Sceloporus occidentalis) was a choice sentinel animal for detecting the presence of I. pacificus nymphs (and larvae) in diverse biotopes even when dragging litter in them was fruitless. The abundance and seasonality of I. pacificus and D. occidentalis adults resembled what had been documented previously for these ticks in northern California. Overall, zero of 27 free-living and 118 lizard-infesting I. pacificus nymphs, 7 (0.29%) of 2392 I. pacificus adults and 2 (0.22%) of 896 D. occidentalis adults were infected with Borrelia burgdorferi sensu lato (Bb sl), but none of them harbored B. miyamotoi, a relapsing-fever group spirochete implicated recently as a zoonotic pathogen in Russia. Borrelia americana and the human pathogen B. burgdorferi sensu stricto were each detected in one (0.04%), and uncharacterized Bb sl in 5 adult I. pacificus (0.21%) that clustered with B. americana. Both PCR-positive D. occidentalis adults contained B. burgdorferi sensu stricto. We conclude that the acarologic risk of being bitten by a B. burgdorferi sensu lato-infected ixodid tick in the habitats studied is slight, which offers a plausible explanation for the low reported incidence of Lyme disease in south coastal California., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The paper reports the prevalence and geographical distribution of Borrelia burgdorferi sensu lato (s.l.) and its genospecies in 12,221 questing Ixodes ricinus ticks collected at 183 locations from all the 41 counties of Romania. The unfed ticks were examined for the presence of B. burgdorferi s.l. by PCR targeting the intergenic spacer 5S-23S. Reverse line blot hybridization (RLB) and restriction fragment length polymorphism (RFLP) analysis were performed for identification of B. burgdorferi genospecies. The overall prevalence of infection was 1.4%, with an average local prevalence between 0.75% and 18.8%. B. burgdorferi s.l. was found in ticks of 55 of the 183 localities. The overall prevalence B. burgdorferi s.l. in ticks in the infected localities was 3.8%. The total infection prevalence was higher in female ticks than in other developmental stages. Three Borrelia genospecies were detected. The most widely distributed genospecies was B. afzelii, followed by B. garinii and B. burgdorferi sensu stricto (s.s.). The study is the first countrywide study and the first report of B. burgdorferi s.s. in Romania. The distribution maps show that higher prevalences were recorded in hilly areas, but Lyme borreliosis spirochetes were also present in forested lowlands, albeit with a lower prevalence., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

We collected 284 ticks in Ethiopia (109 Amblyomma cohaerens, 173 Rhipicephalus decoloratus, and 2 Rhipicephalus praetextatus). We found no rickettsiae and bartonellae. In 7.3% of the A. cohaerens, we found a Borrelia sp. that may represent a new species distant from both relapsing fever group and Lyme borreliae.

Bird migration is a recurring annual and seasonal event undertaken by more than 100 species of birds in the southeast Asian and northeast Palearctic regions that pass through or remain for short periods from April to May and September to November at Hong-do Island, Republic of Korea (ROK). A total of 212 ticks (40 Haemaphysalis flava, 12 H. longicornis, 146 Ixodes turdus, 13 I. nipponensis, and 1 I. ornithophila) were collected from 65/2,161 (3.0%) migratory birds consisting of 21 species that were captured from January, 2008, through December, 2009, as part of the Migratory Birds Center, Hong-do bird banding program for studying bird migration patterns. Adult ticks were assayed individually while larvae and nymphs were pooled (1-22 and 1-6 ticks per pool, respectively) into 31 and 65 pools, respectively. Ticks were assayed for zoonotic pathogens by PCR using 16S rRNA, heat shock protein (groEL), and internal transcribed spacer (ITS) gene primers to amplify genera specific for Anapalsma, Bartonella, and Borrelia PCR amplicons. Using the 16S rRNA-based nested PCR, A. phagocytophilum (n=1) was detected in I. nipponensis collected from Zoothera sibirica and A. bovis (n=1) was detected in I. turdus collected from Emberiza chrysophrys. Borrelia turdi 16S rRNA genes (n=3) were detected in I. turdus and I. nipponensis collected from Turdus pallidus and Zoothera aurea. Borrelia spp. 16S rRNA genes (n=4) were detected in Ixodes ticks collected from Emberiza tristrami, T. pallidus, and Z. aurea. The Bartonella grahamii ITS gene (n=1) was detected by nested PCR assay in I. turdus collected from Z. aurea. These results provide insight into the potential role of migratory birds in the dispersal of ticks and associated tick-borne pathogens throughout their ranges in Asia.

Lyme disease Borrelia spp. are transmitted by Ixodes ticks, and more than 10 species of borreliae have been identified around the world. Recently, another Borrelia sp. has been reported in Asia (Japan, Korea, China, Taiwan and Thailand) as Borrelia valaisiana-related sp. In the present study, we obtained and genetically characterized 19 B. valaisiana-related sp. strains from mammals and ticks. Genetic analyses showed that the Borrelia strains were distinct from B. valaisiana found in Europe. Multilocus sequence typing revealed that these Borrelia isolates formed a monophyletic group with B. yangtze strains in China. Some of the strains were isolated from the bladders of small mammals, and also two strains were experimentally confirmed to be infectious in C3H/HeN mice. We observed that the Borrelia sp. was maintained in the Ixodes granulatus tick after molting. These results suggested that small mammals and I. granulatus were possible reservoir hosts and the vector tick for the Borrelia sp., respectively. B. valaisiana, originally found in Europe, was transmitted mainly by I. ricinus, and birds were mainly thought to be reservoir hosts. Our results suggested that Japanese isolates of B. yangtze (formerly B. valaisiana-related sp.) were distinguishable from B. valaisiana according to the reservoir host and its vector tick. In this study, we also deposited borrelia strain Okinawa-CW62 into bioresource centers as a reference strain of B. yangtze(=DSM 24625, JCM 17189).

Two regions (Jimma and Dire Dawa) in Ethiopia were investigated for the presence of soft ticks. Although no Ornithodoros spp. ticks were collected during this survey, published records of their existence in Ethiopia were found. An overwhelming infestation of Argas persicus was revealed in a village located adjacent to Dire Dawa. These ticks primarily were feeding on poultry, but were also biting humans. Furthermore, hard ticks were collected from livestock and companion animals in these regions. Collected ticks were assessed for Borrelia by real-time PCR followed by conventional PCR and sequencing to identify species present. A. persicus ticks were found to carry B. anserina in 3 of 40 (7.5%) A. persicus tick pools, whilst hard tick pools yielded 2 of 16 (12.5%) positive for B. theileri. Collectively, these borrelial species and their tick vectors are likely to have an important economic impact of particular relevance to subsistence farmers in Ethiopia., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Rocky Mountain spotted fever, Lyme disease, and ehrlichiosis are tick-borne diseases that are reported annually in Kentucky. We conducted a survey to describe infection prevalence of tick-borne pathogens in Amblyomma americanum and Dermacentor variabilis ticks collected in Kentucky. During 2007-2008, we collected 287 ticks (179 D. variabilis and 108 A. americanum) from canine, feral hog, horse, raccoon, white-tailed deer, and human hosts in six counties in Kentucky. Ticks were screened for Rickettsia spp., Borrelia spp., and Ehrlichia spp. by using polymerase chain reaction. Forty-one (14.3%) ticks (31 A. americanum and 10 D. variabilis) were polymerase chain reaction-positive for a Rickettsia spp. Fourteen (4.9%) ticks (6 A. americanum and 8 D. variabilis) were positive for E. chaffeensis, and 4 A. americanum (1.4%) were positive for E. ewingii. One (0.4%) A. americanum was positive for Borrelia lonestari. Although Rocky Mountain spotted fever is diagnosed in Kentucky, no R. rickettsii was found in ticks in this study.

Birds are capable of transporting ticks and, consequently, tick-borne pathogens over long distances and across geographical barriers such as oceans and deserts. The purpose of this study was to assess the prevalence of Borrelia spp. in ticks transported by birds by using PCR. A total of 9768 northward-migrating passerine birds was examined for ticks at 4 bird observatories along the southern Norwegian coast during their spring migration in 2003-2005. Two of the bird observatories were located on islands where flagging revealed very few or no ticks (Akerøya and Store Færder), while the other 2 were located in areas with established dense tick populations: an island, Jomfruland (>100 ticks per hour of flagging) and a mainland locality, Lista (40 ticks in one hour of flagging). Borrelia spp. were found in 70 (13.6%) of 513 examined Ixodes ricinus nymphs (19 B. afzelii, 38 B. garinii, two B. turdi, and 11 B. valaisiana) and in 14 (8.1%) of 172 examined I. ricinus larvae (ten B. garinii, one B. turdi, and three B. valaisiana). This report is the first to identify B. turdi in Europe. Ticks collected from birds of the genus Turdus (T. merula, T. philomelos, and T. iliacus) had a higher prevalence of Borrelia spp. than ticks from the other passerine genera. Ticks that were cofeeding with a Borrelia-infected tick had an increased probability of being infected with the same Borrelia species. Ticks collected on birds from the south-western locality Lista were less likely to have Borrelia than ticks found on birds from the other, more eastern localities. The Turdus spp. are particularly important, both because they carry many ticks per bird and because ticks carried by these species have a higher prevalence of Borrelia. This higher prevalence may be related to Borrelia infection of the birds or transmission of Borrelia through cofeeding. The prevalence of the different Borrelia species in ticks collected from migratory birds may be related to migration routes., (2010 Elsevier GmbH. All rights reserved.)

Unfed adult Ixodes persulcatus ticks from five regions of Russia were examined to analyze the distribution and diversity of Borrelia miyamotoi. DNA of B. miyamotoi was found in 1.8% of ticks from Leningrad Oblast, 2.9% from Sverdlovsk, 4.5% from Novosibirsk, 2.3% from Irkutsk Oblast, and 2.5% from Khabarovsk Krai. The molecular typing of the B. miyamotoi DNA was based on the partial sequencing of the 16S rRNA, p66, and glpQ genes. The only genetic variant of B. miyamotoi was detected in all samples of ticks collected from these five territories.

Analysis of long-term data on the infection rated of the taxonomic complex Borrelia burgdorferi s. l. in Ixodes persulcatus and I. ricinus from Moscow Region is carried out. More than 14 000 tick specimens were examined by dark-field microscopy and 704 specimens were investigated by PCR. Two borrelia genospicies, B. afzelii (6.3%) and B. garinii (20%) proved the most prevalent. The genospecies could form a mixtinfection (11%). Borrelia miyamotoi, B. valaisiana, and B. burgdorferi s. s. were also found in the studied territory, with the total ratio lesser than 6%. Acarotropism of Borrelia proved 2.4 times higher with respect to I. persulcatus, than to I. ricinus. Average long-term infection rates of these tick species were 27.3 and 11.3% respectively. Spatial variability of the infection rate in ticks significantly exceeds interannual fluctuations with the variation coefficients 66 and 26% respectively.

Tick-borne diseases usually comprise a complex epidemiological and ecological network connecting the vector, pathogen, and a group of host species. Symptoms associated with Lyme disease have been reported in Brazil, but no Borrelia sp. has been definitively related to these events. Here we have identified a B. lonestari/B. theileri-related spirochete DNA in the cattle tick Rhipicephalus (Boophilus) microplus from Brazil. Four hundred R. microplus and 80 Amblyomma cajennense ticks were screened, and only 1 horse-fed R. microplus was infected. A Borrelia sp. 16S rDNA sequence was amplified by polymerase chain reaction (PCR) from the total tick DNA with 99% similarity to B. theileri and B. lonestari. Partial flaB sequence was also obtained, demonstrating 96% similarity to the B. lonestari flagellin gene, and the resultant putative amino acid sequence demonstrated 97% identity to B. lonestari flagellin. Moreover, partial glpQ sequence demonstrated 92% similarity to the B. lonestari gene, with a putative amino acid sequence 90% identical to the B. lonestari glycerophosphodiester phosphodiesterase. Phylogenetic analyses clearly include this Brazilian Borrelia sp., denoted "Borrelia," sp-BR in a group of spirochetes aligned with B. theileri and B. lonestari. Thus, hard tick relapsing fever group spirochetes represent a clade of widespread bacteria and herein we describe the first molecular identification of a Borrelia sp. in South America.

Unfed nymphs of Ixodes ricinus were collected from vegetation in a forest on the outskirts of Berlin, Germany and were analyzed for host and pathogen DNA. Pathogens were detected in 47% of the ticks. Borrelia afzelii was the commonest pathogen detected, followed by Rickettsia helvetica. Other pathogens included B. valaisiana, B. garinii, B. burgdorferi sensu stricto, Anaplasma phagocytophilum, and a relapsing fever-like Borrelia. Most of the host DNA detected was of rodent origin and was associated with infection by B. afzelii, R. helvetica, and A. phagocytophilum. Bird DNA was associated with B. valaisiana and B. garinii, and ruminant DNA with A. phagocytophilum. B. afzelii was also found in two ticks that contained bird DNA.

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.

Borrelia lonestari is considered a putative agent of southern tick-associated rash illness (STARI) and is known to occur naturally only in lone star ticks (Amblyomma americanum) and white-tailed deer (Odocoileus virginianus). We used a low passage isolate of B. lonestari (LS-1) to inoculate white-tailed deer, C3H mice, Holstein cattle, and beagles. Animals were monitored via examination of Giemsa and acridine orange stained blood smears, polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) test, and/or culture isolation. Spirochetes were visualized in blood smears of both deer on days post-inoculation (DPI) 6, 8, 12 and one deer on DPI 15. Whole blood collected from deer tested PCR positive starting on DPI 4 and remained positive as long as DPI 28. Both deer developed antibody titers of >64, with a maximum IFA titer of 1024. The organism was reisolated from the blood of both deer on DPI 6 and one deer on DPI 12. All isolation attempts from mice, calves, or dogs were negative, although one of seven mice was transiently PCR positive. Mice and dogs developed an IFA titer > or =64, while calves lacked a detectable antibody response. These preliminary experimental infection trials show that white-tailed deer are susceptible to infection with B. lonestari and develop a spirochetemia following needle-inoculation, while C3H mice, calves, and dogs do not. Results suggest that deer may serve as a vertebrate reservoir host. Tick transmission studies are needed to confirm that this organism can be maintained in a natural cycle involving deer and A. americanum.

Prevalence of infection by Borrelia burgdorferi s.l. and spotted fever group (SFG) rickettsiae was estimated in host-seeking ticks in an area in Tuscany, central Italy, where Lyme borreliosis was reported in a forestry worker. B. burgdorferi s.l. was identified by polymerase chain reaction in 16.7% (95% CI = 10.3, 24.8) of Ixodes ricinus (L.) nymphs and 39.6% (95% CI = 26.5, 54.0) of adults. Borrelia lusitaniae accounted for 82.9% of positive samples, followed by Borrelia garinii (9.8%), Borrelia afzelii (2.4%), and Borrelia burgdorferi s.s. (2.4%). One Rhipicephalus spp. adult was infected with B. garinii (prevalence = 8.3%; 95% CI = 0.21, 38.5). Prevalence of infection by SFG rickettsiae was 38.5% (95% CI = 26.7, 51.4) in I. ricinus nymphs, 34.6% (95% CI = 22.0, 49.1) in I. ricinus adults, and 50% (95% CI = 21.1, 78.9) in Rhipicephalus spp. adults. Phylogenetic analysis showed the similarity of B. lusitaniae strains that were identified in this study and of a strain that was previously isolated from a human patient in Portugal. Results of this study confirm the dominance of B. lusitaniae in areas in the Mediterranean basin and the infection by SFG rickettsiae in I. ricinus.